The protein product of a gene (inaZ) responsible for ice nucleation by Pseudomonas syringae S203 has been identified and purified after overexpression in Escherichia coli. The amino acid composition and the N-terminal sequence of the purified, denatured protein corresponded well with that predicted from the sequence of the inaZ gene. The product of inaZ was also found to be the major component in preparations of ice-nucleating, proteinaceous particles, obtained after extraction with and gel filtration in a mixture of urea and the nondenaturing detergent octyl 1-D-thioglucopyranoside. The activity of these preparations in the absence of added lipid implies that the protein participates directly in the nucleation process.Some bacteria of the genera Pseudomonas, Erwinia, and Xanthomonas can nucleate the crystallization of ice from supercooled water (1-3). Genes encoding the ice-nucleation active (Ina') phenotype have been cloned from Erwinia herbicola, Pseudomonas syringae, and Pseudomonas fluorescens (4-7). Theoretical considerations (8), as well as the actual sequence of the inaZ gene from P. syringae (6), suggest that the bacteria synthesize a template for ice-crystal formation, rather than an enzyme. The translation product predicted from the inaZ sequence is a protein with repetitive primary structure; its tertiary structure, which might also be repetitive, could provide considerable insight into the mechanism of ice nucleation. The ice nuclei of P. syringae are associated with its outer membrane (9) and are believed to contain both protein (9-11) and lipid (11) components. One report has suggested that the inaZ product is a phosphatidylinositol synthase, and that the lipid phosphatidylinositol is a key component of the water-binding template (12).Here, we argue that the active component of bacterial ice nuclei is a protein, the product of inaZ. We have reached this conclusion after purifying the InaZ protein. To Ice-Nucleation Spectra. Ice-nucleation frequencies were measured by a drop-freezing method with an instrument constructed as described by Vali (16). For analysis of column fractions, 20 drops of 10 ,u1 per dilution were examined at a dilution interval of 10-2; in all other cases, 40 drops of 10 p.1 per dilution were tested, with a dilution interval of 10-1. All spectra of subcellular fractions were normalized to the frequency per cell, by dividing the frequency per ml value for a given sample by the ratio (volume of sample)/(number of cells used to prepare sample).Construction of pMWS10. The region of DNA encoding the P. syringae S203 inaZ gene (6) was digested with restriction enzymes Aha III (cutting at nucleotide 775) and EcoRI (cutting at nucleotide 4453), resulting in a fragment beginning 23 base pairs 5' of the initiator codon. The EcoRI end was converted to a HindIII end by addition of a linker, and the fragment was inserted into pKK223.3 (17), so that inaZ was placed downstream of the tac promoter. The construct retained the original ribosome binding site of inaZ. The plasmid pMWS10 ...

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Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations ofP. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.The ability of some species of gram-negative bacteria from the genera Pseudomonas (1, 2, 21, 22), Erwinia (19), and Xanthomonas (23) to nucleate the crystallization of ice demonstrates a unique manipulation of the environment by bacteria. Such bacteria are the major nucleating agents found on the leaves and flowers of many plants (16,17,20) and initiate much of the damage done to crops by frost. The ice nucleation-active (Ina') phenotype may confer a selective advantage on bacteria which express it; whether this advantage exists has been the subject of speculation (17,18 formed E. coli (32); these nuclei are active only at temperatures below -8°C.The precise relationship between ice nucleation proteins and the Ina' phenotype remains unclear. Many models for the role of ice nucleation proteins have been proposed (3,14,29). Such models are premature when two basic questions remain unanswered. Are ice nucleation proteins detectable in the organisms which are sources of ice nucleation genes, and are these proteins present only if the Ina' phenotype is present? This work reports results obtained with a polyclonal antibody raised against one ice nucleation protein. This antibody was used to answer the questions posed above, to identify two previously uncharacterized ice nucleation proteins, and to discover phenomena which indicate that the secondary structure of at least one nucleation protein is periodic.MATERIALS AND METHODS Chemicals, bacterial strains, and culture conditions. All chemicals were reagent grade, purchased from Sigma Chemical Co. unless otherwise note...

The beta-alkyl substituted acrolein congeners crotonaldehyde, trans-2-pentenal, trans-2-hexenal, 2,4-hexadienal, and trans-2-heptenal were clearly mutagenic in a slightly modified preincubation Ames test with Salmonella typhimurium TA100 with and without S9 mix using a threefold bacterial cell density and a 90-min preincubation time, whereas trans-cis-2,6-nonadienal did not show any mutagenic activity. The greatest impediment to adequate mutagenicity testing of these compounds is their toxicity toward bacteria. Within the congener family tested, toxicity increases as a function of both chain length and lipophilicity, and it becomes more and more difficult to demonstrate mutagenicity. Mutagenicity decreases with increasing chain length. This effect may be explained by increasing toxicity. The effect of S9 mix seems to be mostly nonenzymatic detoxication by nonspecific scavanger protection of bacterial cytotoxicity. No indication could be found that bioactivation plays a role in S9-mediated reduction of bacterial cytotoxicity. Although positive mutagenic outcomes could be obtained with the SOS chromotest for other alpha, beta-unsaturated carbonyl compounds, these acrolein congeners were not genotoxic in this test, most probably because they are toxic for the Escherichia coli bacteria PQ37 and PQ243.

The reaction of the alpha, beta-unsaturated ketones methyl vinyl ketone (MVK) and ethyl vinyl ketone (EVK) with nucleosides and 5'-mononucleotides was studied. The genotoxic activity of MVK and EVK in the SOS Chromotest was investigated. Three different types of adducts with deoxyguanosine were found and their structures elucidated: the cyclic 1,N2 adducts, the linear N7 adducts with one still-unreacted carbonyl function, and the cyclic 1,N2, linear N7, bis adducts. The spectroscopic and other relevant characterization data for the deoxyguanosine adducts and the corresponding guanine adducts are presented here together with details of the chromatographic methods used for isolation. The adducts described could also be isolated in the reactions of MVK and EVK with 2'-deoxyguanosine 5'-monophosphate. No adducts could be isolated either with nucleosides other than deoxyguanosine or with nucleotides other than 2'-deoxyguanosine 5'-monophosphate, indicating that the guanine moiety is the most reactive DNA constituent for MVK and EVK. MVK and EVK were clearly genotoxic in the SOS Chromotest according to the criteria of Quillardet and Hofnung. The formation of these adducts was proposed as the mechanism for the genotoxicity of MVK and EVK: all data available support the assumption that MVK and EVK represent a mutagenic and carcinogenic risk for mankind.